A number of high molecular weight carbohydrates are polymers of glucose in which the glucose units are joined by either alpha-1,6-glucosidic linkages or alpha-1,4-glucosidic linkages. It is of considerable industrial importance to be able to cleave these linkages thereby breaking the large carbohydrate molecules into smaller molecules which are more useful in various applications. The breaking of the glucosidic linkages is frequently carried out by enzymes which are produced by microorganisms.
One group of enzymes known as alpha-amylases cleave the alpha-1,4-glucosidic linkages. The alpha-amylase enzymes are produced by such organisms as Bacillus licheniformis and Bacillus stearothermophilus. Such enzymes generally do not cleave the alpha-1,6-glucosidic linkages.
Another class of enzymes, sometimes referred to as glucoamylases, are capable of cleaving both alpha-1,6- and alpha-1,4-glucosidic linkages. These enzymes remove one glucose unit at a time from the nonreducing end of the large carbohydrate molecule. While they are capable of hydrolyzing certain alpha-1,6-glucosidic linkages, they hydrolyze the alpha-1,4-glucosidic linkages much more rapidly.
Other enzymes which hydrolyze certain alpha-1,6-linkages are classified as pullulanases. These enzymes are capable of hydrolyzing the alpha-1,6-linkages in the polysaccharide, pullulan, to give the trisaccharide, maltotriose. They do not hydrolyze the alpha-1,4-linkages in pullulan. The first pullulanase described was an extracellular enzyme produced by Aerobacter aerogenes. References to this enzyme and other enzymes capable of hydrolyzing alpha-1,6-linkages are given in U.S. Pat. Nos. 3,897,305 and 3,992,261. These enzymes are thermolabile and cannot be used at temperatures much above 50.degree. C. A pullulanase enzyme produced by the bacterium, Bacillus acidopullulyticus, is described in British Patent 2,097,405. This pullulanase is sufficiently thermostable to be employed at 60.degree. C. These enzymes, as well as all previously-known pullulanases, have been obtained from aerobic microorganisms.
Recently, it has been discovered that the anaerobic microorganism, Thermoanaerobium brockii (hereafter written T. brockii), produces a pullulanase enzyme with even greater thermostability. We have now isolated the gene coding for this thermostable pullulanase and inserted it into plasmid vectors. These vectors have in turn been incorporated into strains of E. coli and B. subtilis. The resulting genetically-engineered microorganisms produce the thermostable pullulanase when they are grown in suitable media.